CN103436599B - LAMP detection primer group of NPT II marker screening gene, kit and detection method - Google Patents
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Abstract
The invention relates to an LAMP detection primer group of an NPT II marker screening gene, a kit and a detection method, wherein the LAMP detection primer group includes the following primers of an outer primer F3: CTCGACGTTGACTCTGATG, an outer primer B3: TGATGCTCATCGTCCAGT, an inner primer FIP: TAGCCGGTTCATGCGTAT GCTCATCTCACCTTGCACCT, an inner primer BIP: CCTATCGTCCACCTTGCGACTTC CAGTTCGACTTGACC, a loop primer FLP: TTGCATCTGCCATGTAGGTTA and a ring primer BLP: CGTTCACGGTTGGATGCC. The LAMP detection primer group provided by the invention has good specificity for the NPT II marker screening gene.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of food safety, relates to a loop-mediated isothermal amplification technology, and particularly relates to an LAMP (loop-mediated isothermal amplification) detection primer group, a kit and a detection method for transgenic crops containing NPTII (NPTII marker screening genes).
[ background of the invention ]
Transgenic crops refer to plants and their progeny produced by integrating exogenous genes into the genome of recipient plants and changing the genetic composition thereof by recombinant DNA technology, and are also called Genetically Modified Organisms (GMOs). Since the first transgenic plant appeared in 1983, the plant area and sales revenue of transgenic crops worldwide increased by multiples. However, in a serious scientific sense, the biological safety of the transgenic agricultural products has no definite conclusion at present. Although China stipulates that all transgenic crops and byproducts thereof should be marked since 3, 20 and 3 months in 2002, agricultural products injected with transgenic characters are still rarely seen under the situation that the number of transgenic crops is increasing. Therefore, the development of simple transgenic soybean detection technology and the detection of related products are imperative.
At present, the transgenic crops are generally detected by using a DNA-based detection technology. Such techniques mainly include traditional qualitative PCR and quantitative PCR. These detection techniques all require a special DNA amplification apparatus (PCR apparatus) to be able to perform. Loop-mediated isothermal gene AmpliFication (Loop-mediated isothermal AmpliFication, LAMP) relies on a primer capable of recognizing 6 specific regions on a target sequence and a DNA polymerase with strand displacement properties, and the DNA polymerase is maintained at a constant temperature for tens of minutes by using the strand displacement DNA polymerase to complete a nucleic acid AmpliFication reaction; the target gene DNA fragment can be amplified 109-fold and 1010-fold within 1 hour, and whether the amplification is carried out can be identified only by observing the white turbid precipitate with naked eyes, without the need for an electrophoretic detection process.
The sequence information of the NPTII marker screening gene is known by referring to the relevant literature of the transgenic soybean line MON89788, and the partial sequence information specifically comprises the following steps:
ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTG GAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGAT GCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAA GACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGG CTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTT GTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGC AGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGG CTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCG ACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCC AGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGATGATC TCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATG GCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCT ATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGC AGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGA。
[ summary of the invention ]
The first technical problem to be solved by the invention is to provide an LAMP detection primer group of NPTII marker screening genes, which has good specificity to the NPTII marker screening genes.
The second technical problem to be solved by the invention is to provide an LAMP detection kit for NPTII marker screening genes, which is convenient for LAMP detection of the NPTII marker screening genes.
The third technical problem to be solved by the invention is to provide the LAMP detection method for the NPTII marker screening gene, which has the characteristics of high sensitivity, good specificity, quick and reliable detection and simple operation.
The first technical problem is achieved by the following technical solutions:
an LAMP detection primer group of NPTII marker screening genes is characterized by comprising the following primers:
outer primer F3: CTCGACGTTGACTCTGATG, respectively;
outer primer B3: TGATGCTCATCGTCCAGT, respectively;
inner primer FIP:
TAGCCGGTTCATGCGTATGCTCATCTCACCTTGCACCT；
the inner primer BIP:
CCTATCGTCCACCTTGCGACTTCCAGTTCGACTTGACC；
loop primer FLP: TTGCATCTGCCATGTAGGTTA, respectively;
loop primer BLP: CGTTCACGGTTGGATGCC are provided.
Experiments prove that the DNAs of the LAMP detection primer group provided by the invention have no non-specific amplification. Therefore, the LAMP detection primer group provided by the invention has good specificity to the NPTII marker screening gene.
The second technical problem is achieved by the following technical solutions:
the LAMP detection kit for NPTII marker screening genes is characterized by comprising the following components:
(ii) a Wherein each primer in the primer solution corresponds to each primer in the LAMP detection primer group of the NPTII marker screening gene.
Also comprises a color developing agent; the color developing agent is a fluorescent dye SYBR Green I.
Controls were also included: the positive control is DNA solution of corn flour containing NPTII marker screening gene with purity of 10% and concentration of 100ng/ul, and the negative control is ddH2O。
The LAMP detection kit provided by the invention integrates the LAMP detection primers and related materials for LAMP detection, and is convenient for a user to carry out LAMP detection on NPTII marker screening genes.
The third technical problem is realized by the following technical scheme:
the LAMP detection method of the NPTII marker screening gene is characterized by comprising the following steps:
(101) extracting a template DNA to be detected from a sample to be detected;
(102) performing a loop-mediated isothermal gene amplification reaction:
configuring an LAMP reaction system, wherein the LAMP reaction system with the volume of each 25 mu L specifically comprises the following steps:
placing the LAMP reaction system in an LAMP turbidimeter for constant temperature reaction at 63 ℃ for 60min, and then preserving heat at 80 ℃ for 5 min; wherein each primer in the primer solution corresponds to each primer in the LAMP detection primer group of the BAR gene.
Experiments prove that the LAMP detection method provided by the invention combines the good specificity of the primers and the advantages of the LAMP method, and has the following characteristics: the method has the advantages of high sensitivity, rapidness, reliability, good stability, simple operation and simple identification.
[ description of the drawings ]
FIG. 1 is a diagram showing the results of the LAMP reaction carried out in example one;
FIG. 2 is a graph showing the results of the sensitivity test conducted in example II;
FIGS. 3 to 6 are graphs showing the results of the specificity test conducted in example III;
FIGS. 7 to 9 are graphs showing the results of stability experiments conducted in example four.
[ detailed description ] embodiments
Example one
The LAMP detection primer group for NPTII marker screening genes provided by the embodiment comprises the following primers:
outer primer F3: CTCGACGTTGACTCTGATG, respectively;
outer primer B3: TGATGCTCATCGTCCAGT, respectively;
inner primer FIP:
TAGCCGGTTCATGCGTATGCTCATCTCACCTTGCACCT；
the inner primer BIP:
CCTATCGTCCACCTTGCGACTTCCAGTTCGACTTGACC；
loop primer FLP: TTGCATCTGCCATGTAGGTTA, respectively;
loop primer BLP: CGTTCACGGTTGGATGCC are provided. .
The kit for screening genes by NPTII markers provided by the embodiment comprises the following components:
(ii) a Wherein each primer in the primer solution corresponds to each primer in the LAMP detection primer group of the BAR gene.
The method for detecting a sample to be detected provided by the embodiment specifically includes the following steps:
(101) extracting a template DNA to be detected from a sample to be detected;
(102) performing a loop-mediated isothermal gene amplification reaction:
configuring an LAMP reaction system with the total volume of 25 mu L, which specifically comprises the following steps:
(ii) a Wherein, each primer in the primer solution corresponds to each primer in the LAMP detection primer group of the BAR gene; placing the LAMP reaction system in an LAMP turbidimeter for carrying out constant temperature reaction at 63 ℃ for 60min, and then carrying out heat preservation at 80 ℃ for 5 min;
(103) the result judgment can be carried out in one of the following two ways:
the first mode is as follows: judging an amplification result by observing the turbidity change of the sediment in the reaction tube, wherein if the sediment is positive, the amplification result is negative if the sediment is not positive;
the second way is: 1-2. mu.L of a color-developing agent (usually 2. mu.L) was added to each reaction tube, and mixed well, and the mixture was positive in green and negative in orange.
Selecting the primer for LAMP detection, wherein the primer has the characteristics of early peak time and strong signal, and replacing the template DNA to be detected with a DNA solution of corn flour containing NPTII marker screening genes as shown in figure 1, and performing the step (102) twice; by ddH2O as a negative control, in particular ddH2Replacing the template DNA to be detected with O, and performing the step (102) for two times; the results of the four LAMP reactions are shown in FIG. 1, the CH1 line and CH2 line correspond to ddH2The O, CH3 line and CH4 line correspond to DNA solutions of corn meal containing NPTII marker selection genes.
In this context, the parameters of the DNA solution of corn meal containing the NPT II marker selection gene are: the purity was 5% and the DNA concentration was 100 ng/ul.
In this context, the determination of the DNA concentration and purity is:
adding ddH into 5 mu LDNA solution2Diluting to 1mL in an O gradient, and measuring optical density values at 260nm and 280nm by using a nucleic acid protein analyzer or an ultraviolet spectrophotometer; the concentration of DNA was calculated according to the following formula:
C＝A×N×50/1000；
wherein C-DNA concentration (. mu.g/. mu.L), A-absorbance at 260nm, N-nucleic acid dilution factor, 1OD260nm50 μ g/mL double-stranded DNA; when OD is reached260/OD280When the ratio is between 1.7 and 1.9, the kit is suitable for LAMP detection.
Example two
In this example, the sensitivity experiment is mainly performed on the detection method provided in the first embodiment, specifically:
the DNA solution (with the purity of 10%) of the transgenic corn MON863 containing the NPTII marker screening gene is respectively diluted into five mixed solutions with different purities by using the DNA solution of the non-transgenic rice, and in the five mixed solutions, the purities of the DNA of the transgenic corn MON863 are respectively 5%, 1%, 0.5%, 0.1% and 0.05%; taking 2 mu L of each mixed solution respectively, and replacing the template DNA to be detected by the step (102) described in the first operation example; by ddH2O as a negative control, and a DNA solution of corn meal containing NPTII marker screening gene as a positive control, respectively replacing the template DNA to be detected, and the step (102) described in the first working example.
In this context, the parameters of the DNA solution of transgenic maize MON863 containing the NPTII marker selection gene are: the purity was 10% and the concentration was 100 ng/. mu.l.
The parameters of the DNA solution of corn meal containing the NPTII marker selection gene are: the purity was 10% and the concentration was 100 ng/. mu.l.
The results of the experiment are shown in FIG. 2, lines CH 21-CH 28 correspond to DNA solution of Zea mays flour containing NPTII marker selection gene, ddH, respectively2O, DNA solution (purity: 10%) of transgenic corn MON863 containing NPTII marker screening gene, mixed solution with purity of 5%, mixed solution with purity of 1%, mixed solution with purity of 0.5%, mixed solution with purity of 0.1% and mixed solution with purity of 0.05%.
As can be seen from fig. 2, the detection limit of the detection method of the first embodiment can be achieved by detecting the NPTII marker screening gene in the DNA solution (containing the NPTII marker screening gene) of the transgenic corn MON863 with the purity of 0.5%; the reaction time can be determined to be 45min, with a peak at the lowest detection limit of about 23min and no false positive. Therefore, the method has higher sensitivity.
EXAMPLE III
In this example, the specific experiment is mainly performed on the primers provided in the first embodiment, specifically:
replacing the DNA of the template to be detected with the DNA of mung bean, broad bean, pig, oat, orange, sheep, duck, potato, honey pear, grass shrimp, chickpea, chicken, goose, rice, white kidney bean, cowpea, buckwheat, tomato, almond, walnut and donkey respectively (102) in the first operation example; by ddH2O as a negative control, and a DNA solution of corn meal containing NPTII marker screening gene as a positive control, respectively replacing the template DNA to be detected, and the step (102) described in the first working example.
The results of part of the experiment are shown in FIG. 3, in which case the lines CH 31-CH 38 correspond to DNA solution of Zea mays meal containing NPTII marker selection gene, ddH, respectively2O, mung bean, broad bean, pig, oat, orange and sheep.
The results of some experiments are shown in FIG. 4, the CH41 line-CH 48 line corresponds to duck, potato, honey pear, grass shrimp, chickpea, chicken, goose and rice, respectively.
Part of the experimental results are shown in FIG. 5, the CH51 line-CH 58 corresponds to white kidney bean, cowpea, buckwheat, tomato, almond, walnut and donkey, respectively.
The results of some of the experiments are shown in FIG. 6, where in FIG. 6, the CH61 line-CH 68 corresponds to: MON863, DP356043, GA21, MON89788, BT11 containing NPTII marker selection gene, EH92-527-1, MON88017, MON810 containing NPTII marker selection gene.
As can be seen from the results shown in FIGS. 3 to 6, the primers have specificity only for the NPTII marker screening gene, and have no amplification for other transgenic lines and non-transgenic species not containing the NPTII marker screening gene, so that the specificity of the primers is good, and therefore, the detection method provided by the invention is very reliable on the premise that the specificity of the primers is high.
Example four
In this example, the stability test is mainly performed on the detection method provided in the first embodiment, and specifically includes:
diluting DNA solution (with the purity of 10%) of transgenic corn MON863 containing NPTII marker screening gene with non-transgenic corn DNA to the purity of 5%, respectively taking 20 parts of the diluted DNA solution, and replacing the template DNA to be detected (102); by ddH2O as a negative control and a DNA solution of corn meal containing NPTII marker screening gene as a positive control, respectively replacing the template DNA to be detected and carrying out the step (102) described in the first embodiment.
The results are shown in FIGS. 7 to 9, where the curves CH73-CH78 in FIG. 7, 8 curves in FIG. 8, and 6 curves in FIG. 9 correspond to the 20 diluted DNA solutions, and the lines CH71 and CH72 in FIG. 7 correspond to the DNA solution of corn meal containing NPTII marker screening gene, ddH, respectively2O; from the figure, the appearance positions of the 20 curves are very close, so that the detection method has good stability.
The present invention is not limited to the above-described embodiments, and simple substitutions based on the above-described embodiments, which are not inventive, should fall within the scope of the present disclosure.
Sequence listing
<110>
LAMP detection primer group, kit and detection method for <120> NPTII marker screening gene
<130> Tang Ding Shi Ming; qian Zheng Jie; feng jia inspection
<160>6
<170>PatentIn version3.5
<210>1
<211>19
<212>DNA
<213> Artificial sequence
<400>1
CTCGACGTTGACTCTGATG 19
<210>2
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<212>DNA
<213> Artificial sequence
<400>2
TGATGCTCATCGTCCAGT 18
<210>3
<211>38
<212>DNA
<213> Artificial sequence
<400>3
TAGCCGGTTCATGCGTATGCTCATCTCACCTTGCACCT 38
<210>4
<211>38
<212>DNA
<213> Artificial sequence
<400>4
CCTATCGTCCACCTTGCGACTTCCAGTTCGACTTGACC 38
<210>5
<211>21
<212>DNA
<213> Artificial sequence
<400>5
TTGCATCTGCCATGTAGGTTA 21
<210>6
<211>18
<212>DNA
<213> Artificial sequence
<400>6
CGTTCACGGTTGGATGCC 18
Claims (6)
- An LAMP detection primer group of NPTII marker screening genes is characterized by comprising the following primers:outer primer F3: CTCGACGTTGACTCTGATG, respectively;outer primer B3: TGATGCTCATCGTCCAGT, respectively;inner primer FIP:TAGCCGGTTCATGCGTATGCTCATCTCACCTTGCACCT；the inner primer BIP:CCTATCGTCCACCTTGCGACTTCCAGTTCGACTTGACC；loop primer FLP: TTGCATCTGCCATGTAGGTTA, respectively;loop primer BLP: CGTTCACGGTTGGATGCC are provided.
- The LAMP detection kit for NPTII marker screening genes is characterized by comprising the following components:(ii) a Wherein,outer primer F3: CTCGACGTTGACTCTGATG, respectively;outer primer B3: TGATGCTCATCGTCCAGT, respectively;inner primer FIP:TAGCCGGTTCATGCGTATGCTCATCTCACCTTGCACCT：the inner primer BIP:CCTATCGTCCACCTTGCGACTTCCAGTTCGACTTGACC；loop primer FLP: TTGCATCTGCCATGTAGGTTA, respectively;loop primer BLP: CGTTCACGGTTGGATGCC are provided.
- 3. The LAMP detection kit for NPTII marker screening genes according to claim 2, characterized by further comprising a color developing agent.
- 4. The LAMP detection kit for NPTII marker screening genes as claimed in claim 3, wherein the color-developing agent is a fluorescent dye SYBR Green I.
- 5. The LAMP detection kit for NPTII marker screening gene according to claim 2, characterized by further comprising a control: the positive control is DNA solution of transgenic corn MON863 containing NPTII marker screening gene with the purity of 10% and the concentration of 100ng/ul, and the negative control is ddH2O。
- The LAMP detection method of NPTII marker screening gene is characterized by comprising the following steps:(101) extracting a template DNA to be detected from a sample to be detected;(102) performing a loop-mediated isothermal gene amplification reaction:configuring an LAMP reaction system, wherein the LAMP reaction system with the volume of each 25 mu L specifically comprises the following steps:placing the LAMP reaction system in an LAMP turbidimeter for constant temperature reaction at 63 ℃ for 60min, and then preserving heat at 80 ℃ for 5 min; wherein,outer primer F3: CTCGACGTTGACTCTGATG, respectively;outer primer B3: TGATGCTCATCGTCCAGT, respectively;inner primer FIP:TAGCCGGTTCATGCGTATGCTCATCTCACCTTGCACCT；the inner primer BIP:CCTATCGTCCACCTTGCGACTTCCAGTTCGACTTGACC；loop primer FLP: TTGCATCTGCCATGTAGGTTA, respectively;loop primer BLP: CGTTCACGGTTGGATGCC are provided.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1470649A (en) * | 2002-07-26 | 2004-01-28 | 深圳市匹基生物工程股份有限公司 | Primer sequence for NptII gene containing transgenic crop nucleic acid amplification |
| CN102559897A (en) * | 2012-01-13 | 2012-07-11 | 广州华峰生物科技有限公司 | Primer group for NPT(Noctumal Penile Tumescence)II gene detection, corresponding reagent kit for and use method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1470649A (en) * | 2002-07-26 | 2004-01-28 | 深圳市匹基生物工程股份有限公司 | Primer sequence for NptII gene containing transgenic crop nucleic acid amplification |
| CN102559897A (en) * | 2012-01-13 | 2012-07-11 | 广州华峰生物科技有限公司 | Primer group for NPT(Noctumal Penile Tumescence)II gene detection, corresponding reagent kit for and use method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 环介导等温扩增核酸技术及其应用;匡燕云等;《微生物学通报》;20071231;第34卷(第3期);557-560 * |
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